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Development of a Multistrain CRISPR System for HIV-1 Elimination Session: P-E07

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Development of a Multistrain CRISPR System for HIV-1 Elimination Session: P-E07 Poster: 00353 Development of a Multistrain CRISPR System for HIV-1 Elimination Session: P-E07 Jonathan Herskovitz1, Mahmudul Hasan1, Wilson Blomberg1, Milankumar Patel1, Maxim Oleynikov2, Jatin Machhi1, Insiya Mukadam1, Benson Edagwa1, Kamel Khalili3, Bhavesh Kevadiya4, Channabasavaiah B. Gurmumurthy1, and Howard E. Gendelman1 1University of Nebraska Medical Center, Omaha, NE 2Cornell University, Ithaca, NY 3Temple University Medical Center, Philadelphia, PA 4Stanford University School of Medicine, Stanford, CA Abstract CombineD Tat/Rev/gp41 gRNAs HIV-1 VLPs target CD4 Background: A key challenge to eliminating HIV-1 infection rests in removing the A diverse array of integrated HIV-1 strain DNA from the genomes of CD4+ inactivate multiple HIV-1 strains Day 0 Day 1 Day 7 Day 10 leukocytes. A means of achieving such lies in newer CRISPR-Cas9 technologies. Identifying a gRNA system to target the multitude of HIV-1 proviral DNA A Infection Virus Washout Treatment Harvest HIV-1 sequences and a delivery vector for CD4-specific delivery remain challenges. NL4-3eGFP 1. VLP (2 ng p24) 1. DNA (PCR) MOI 0.05 2. Pellet (Flow) 2. VLPCRISPR (2 ng p24) Purpose: To develop a CRISPR-Cas9 system with broad abilities to cleave 3. pCRISPR (1 μg DNA) integrated HIV-1 proviral DNA delivered in CD4-targeted virus-like particles ± gp120 (1 μg) (VLPs). B C Hypothesis: CRISPR-Cas9 inactivation of the HIV-1 transactivator of transcription BanD 2 AliGnment (tat) gene in conserved subgenomic regions could reduce or eliminate viral replication. When loaded in HIV-1 envelope-pseudotyped VLPs, CRISPR-Cas9 could excise HIV-1 DNA in CD4+ T cells and macrophages in tissue viral reservoirs. Methods: CRISPR-Cas9 guide RNAs (gRNAs) designed against HIV-1 tat, were developed against known HIV-1 sequences with limited to no off-target loci. The gRNAs were cloned into an spCas9 expression vector and screened for antiretroviral activity against seven HIV-1 strains. LTR- and gag- gRNAs served B C as reference controls. Delivery of CRISPR-Cas9 to CD4+ leukocytes in VLPS BanD 3 AliGnment was assessed. Results: Tat-directed gRNAs that also cleave rev (and env) exhibit greatest reduction in RT-activity in a 48-hour co-transfection screen. Reduction in RT- activity correlate with % sequence conservation. When delivered in VLP format, LTR/gag mediated excision was shown to be gp120::CD4 specific. TarGetinG HIV-1tat D Figure 3. Screen of tat-directed gRNA reduction in RT-activity. A transfection screen of gRNAs cloned into spCas9 expression plasmid pX333 (Addgene #64073) was performed in HEK293FT cells co-transfected with HIV-1 encoding plasmids. LTR-1 and GagD were both cloned onto the same plasmid. After 48 hours, supernatant was Figure 1. CRISPR-Cas9 gRNAs. Guide RNAs were designed against various positions of HIV-1 tat, tat/rev, or assessed for reverse-transcriptase (RT) activity (A). Percent reduction from untreated controls is represented in tat/rev/env are shown in green. Control gRNAs against HIV-1 LTR and gag previously demonstrated to show heat-map form (B). DNA was extracted from cells co-transfected with pHIVNL4-3 and assayed by PCR for excision antiretroviral effects were also included in these studies as reference controls (red). of the first exon of tat by dual tat-directed gRNAs (C). CPM: counts per minute 3H-thymidine incorporation. Figure 5. VLPs for CRISPR-Cas9 delivery and viral excision. CD4+ T cells were infected with HIV-1 for 7 days then treated with HIV-1envelopef VLPs (2 ng HIV-1 p24 / 106 cells) or appropriate controls for 72 hours according the timeline depicted in (A). As a control for specificity to CD4+ T cells, excess recombinant HIV-1gp120 (1 μg / 106 cells) was added during treatment. (B) PCR for CRISPR-Cas9-mediated excision of 5’ LTR and gag sequences. (C) PCR amplicons outlined in (B) were extracted, Sanger sequenced, and aligned using NCBI BlastN. (D) gRNA Sequence Conservation gRNAs and HIV-1 suppression Summary alignment charts for sequenced DNA amplicons. VLP: virus-like particles synthesized by pHIV89.6env, pCRISPRLTR-Gag, and psPAX2 co-transfection. % Sequence Conservation gRNA Name CRISPR-OFF Score (n = 4004 HIV-1 Strains) LTR-1 (control) 27.12 % 6.937 Conclusions GagD (control) 11.14 % 4.071 TatG 67.15 % 3.803 1. HIV-1 tat gRNAs can maintain >50% sequence conservation. TatD 59.04 % 6.284 2. TatD and TatE CRISPR-Cas9 reduce progeny virion production TatH 51.12 % 4.662 in diverse HIV-1 molecular clones. TatA 2 29.79 % 7.327 TatI 25.90 % 6.844 3. TatD and TatE cleavage of tat/rev(/env) lead to greatest viral TatE 15.26 % 3.090 suppression. TatF 6.19 % 6.467 4. HIV-1 virus-like particles can delivery CRISPR-Cas9 payloads to TatE 5.82 % 6.021 2 infected CD4+ T cells and macrophages. Figure 2. Sequence conservation and Off-target scores among gRNAs. Tat-directed or control gRNA sequences were screened against 4004 clinical HIV-1 DNA sequences (hiv.lanl.gov) for full sequence matches. AcknowleDGments Off-target cleavage potential of each gRNA against the human genome was assessed using CRISPR-OFF v1.1 Figure 4. gRNA induced RT activity reductions correlate with viral sequence conservations. The average percent RT-activity reduction against seven HIV-1 molecular clones was determined. Pearson correlation was This work was supported in part by NIH Grants R01 AG043530, P01 DA028555, P30 tool (rth.dk/resources/crispr/crisproff), wherein high scores correlate with low off-target DNA breaks. MH062261, R01 MH115860, R01 NS034249, R01 NS036126, T32 NS105594 and the determined to be significant when TatE (a positive outlier) is excluded from metanalysis. Carol Swartz Emerging Neuroscience Laboratory..
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